The disruption of the mouse gene encoding cPLA2 by homologous recombination is currently in progress. Mice deficient in functional cPLA2 will be generated by targeting the region of the gene encoding the active site of the enzyme at serine 228 which is known to be essential for enzymatic activity. Only the cDNA sequence encoding the active site region has been published, and no genomic sequence of this region of the gene is available. Therefore, PCR was used to determine the approximate location of the exon encoding serine 228 and the next exon 3' of this active site exon. Probes corresponding to these two exons were used to screen a Charon 35 genomic library containing DNA from E14TG2a mouse embryonic stem cells. From this library, approximately 15 kb of the cPLA2 gene which includes the active site region was cloned. This clone has been extensively mapped by Southern hybridization and restriction fragments have been subcloned into Bluescript. Additional clones which encode for the serine 228 exon and the region 5' of this exon have been isolated and restriction fragments are being subcloned into Bluescript. The targeting construct will disrupt the cPLA2 gene with the neomycin resistance gene approximately 10 bases 5' of the 3' splice junction of the exon encoding serine 228. The disruption of the cPLA2 gene at this site will eliminate the splice junction and at most permit transcription of an aberrant message with no translation of functional cPLA2.
